The enzymatic dissociation of tissue into individual cells and cell clusters is useful in a wide variety of laboratory, diagnostic and therapeutic applications. These applications involve the isolation of many types of cells for various uses, including microvascular endothelial cells for small diameter synthetic vascular graft seeding, hepatocytes for gene therapy, drug toxicology screening and extracorporeal liver assist devices, chondrocytes for cartilage regeneration, and islets of Langerhans for the treatment of insulin-dependent diabetes mellitus. Enzyme treatment works to fragment extracellular matrix proteins and proteins which maintain cell-to-cell contact. Since collagen is the principle protein component of tissue ultrastructure, the enzyme collagenase has been frequently used to accomplish the desired tissue disintegration.
Different forms of crude bacterial collagenase derived from Clostridium histolyticum are commercially available and are used to dissociate cells and cell clusters from tissue. These crude collagenases, derived from cell culture supernatants, typically contain a mixture of protease enzymes (exhibiting both collagenolytic and non-specific proteolytic activities) and non-protease components (e.g., fermentation by-products, media components, pigments, other enzymes such as phospholipase, and endotoxins).
Analysis of commercially-available crude collagenases has shown extreme variations in the concentration and ratios of the protease and non-protease components. Such compositional variability is reflected as well in the variability and consequent lot-to-lot unpredictability of collagenase product performance in tissue dissociation protocols. In addition to this inherent activity variability, each lot of commercial collagenase loses activity and performance characteristics over time. Finally, in addition to these problems associated with compositional variability, the use of crude collagenases in cell harvest/tissue dissociation protocols usually results in less- than-desired results in terms of recovery of cell viability, cell number and cell function. These problems are particularly significant where the cells are targeted for use in transplantation or for monitoring the impact of effector molecules on cell function. For example, it has been demonstrated that the efficacy of islet transplantation is dependent in part on the mass of islets and their viability. In addition, the drug detoxification function of recovered hepatocytes is significantly impaired by damage to the cells occurring during liver tissue dissociation and cell isolation. For most uses of recovered cells it is critical for optimum cell performance that damage to the recovered cells and cell clusters be minimized.
Skilled practitioners have recognized the importance of the consistent/predictable activity of protease enzymes used in tissue dissociation protocols for efficacious cellular isolation (i.e., maintaining cellular integrity, recovering larger cell clusters and more cells or cell clusters). Specifically, the purity of collagenase compositions and the desirability of the presence of defined amounts of both C. histolyticum collagenase class I (collagenase I) and collagenase class II (collagenase II) enzymes with at least two neutral proteases has been found to influence the efficacy of pancreatic islet isolation. However, there still exists a need for identifying optimized enzyme compositions which provide for rapid dissociation of tissue and recovery of a greater number of viable cells.